Synaptic aging disrupts synaptic morphology and function in cerebellar Purkinje cells

Abstract

Synapses are key structures in neural networks, and are involved in learning and memory in the central nervous system. Investigating synaptogenesis and synaptic aging is important in understanding neural development and neural degeneration in diseases such as Alzheimer disease and Parkinson's disease. Our previous study found that synaptogenesis and synaptic maturation were harmonized with brain development and maturation. However, synaptic damage and loss in the aging cerebellum are not well understood. This study was designed to investigate the occurrence of synaptic aging in the cerebellum by observing the ultrastructural changes of dendritic spines and synapses in cerebellar Purkinje cells of aging mice. Immunocytochemistry, DiI diolistic assays, and transmission electron microscopy were used to visualize the morphological characteristics of synaptic buttons, dendritic spines and synapses of Purkinje cells in mice at various ages. With synaptic aging in the cerebellum, dendritic spines and synaptic buttons were lost, and the synaptic ultrastructure was altered, including a reduction in the number of synaptic vesicles and mitochondria in presynaptic termini and smaller thin specialized zones in pre- and post-synaptic membranes. These findings confirm that synaptic morphology and function is disrupted in aging synapses, which may be an important pathological cause of neurodegenerative diseases.

Keywords: Purkinje cells; aging; cerebellum; degenerative disease; dendritic spine; mice; nerve regeneration; neural regeneration; neurodegenerative diseases; synapse; synaptic ultrastructure; synaptogenesis.

Figure 1

Aging of dendritic spines in Purkinje cells (DiI diolistic assay). (A−C) Purkinje cells at various ages. At P7, the Purkinje cells were not mature. (A) A bold primary dendrite extended from the cell body toward the molecular layer, and the primary dendrite branched into 2−3 secondary dendrites. (B) With increasing age, the dendrites branched repeatedly and the Purkinje cells appeared fan-shaped with numerous dendrites in the molecular layer. At P30, the dendrites could reach up to the pia. (C) At P18M, Purkinje cells had aged and had fewer branched dendrites. (D–I): High magnification images of dendritic spines at various ages. (D) At P3, the spines started to sprout with sparse filopodia on dendrites. (E) At P7, the dendritic spines became short, and their number increased. (F) Dense tidy spines were found at P14. (G) At P30, the number of dendritic spines was maximal. (H) At P12M, the dendritic spines started to gradually decrease. (I) At P18M, many dendritic spines were lost. Scale bars: 30 μm in A−C, 5 μm in D−I. P3, P7, P14, P30: Postnatal 3, 7, 14, 30 days; P12M, P18M: postnatal 12, 18 months.

Figure 2

Purkinje cells and synaptophysin-immunoreactive puncta at various ages (calbindin and synaptophysin immunocytochemistry and DAPI counterstaining). Calbindin-immunoreactive Purkinje cells (red), synaptophysin-immunoreactive presynaptic puncta (green), and DAPI-labeled nuclei (blue) were visualized. At P3, Purkinje cells in the PCL (red) had short processes. The dendrites did not extend into the molecular layer. (A) Synaptophysin-immunoreactive puncta (green) were located around Purkinje cells. At this age, there were almost no synaptophysin-immunoreactive puncta in the molecular layer. (B) At P7, many dendrites extended into the molecular layer, and there was a large increase in the number of synaptophysin-immunoreactive presynaptic puncta around cell bodies, dendrites and spines. At P30, the branched dendrites of Purkinje cells had filled the molecular layer, and the dendrites had reached the pia. (C) Synaptophysin-immunoreactive puncta were very dense around Purkinje cells. After P12M, the synapses showed signs of aging, and the number of synaptic buttons decreased. (D) At P18M, the number of synaptophysin-immunoreactive buttons was at a minimum. Scale bar: 20 μm. P3, P7, P30: Postnatal 3, 7, 30 days; P12M, P18M: postnatal 12, 18 months. ML: Molecular layer; PCL: Purkinje cell layer; IGL: internal granular layer.

Figure 3

Changes in the density of dendritic spines and synaptic buttons in Purkinje cells during synaptic aging. (A) Fitted curve between spine density (Y) and age (X): The regression equation is shown with the formula: Y = 0.025X³ − 0.416X² + 2.172X − 1.586 (R² = 0.989, P < 0.05). The dendritic spines at P12M and P18M were compared with control (P3M). (B) Fitted curve between synaptic buttons (Y) and age (X): The regression equation is shown with the formula: Y = 0.013X³ − 0.244X² + 1.384X − 0.973 (R² = 0.972, P < 0.05). Synaptophysin-immunoreactive puncta at P12M and P18M were compared with control (P3M). *P < 0.05, vs. control (n = 60; one-way analysis of variance followed by Student-Newman-Keuls analysis). P3, P14: Postnatal 3, 14 days; P1M, P3M, P6M, P12M, P18M: postnatal 1, 3, 6, 12 and 18 months.

Figure 4

Synaptic ultrastructure during synaptic development and aging (transmission electron microscope). (A) At P14, synapses had a presynaptic membrane, synaptic cleft and postsynaptic membrane. There were numerous synaptic vesicles in the presynaptic terminal, and obvious thick pre- and post-synaptic membranes. (B) At P30, synapses looked more mature with typical synaptic structure. (C, D) At P18M, the aged synapse had fewer synaptic vesicles and thin pre- and post-synaptic membranes. Arrows: Synapse; arrowheads: synaptic vesicles. Scale bar: 300 nm. P14, P30: Postnatal 14, 30 days; P18M: Postnatal 18 months.